Calpain 10

Calpain 10: a mitochondrial calpain and its role in calcium-induced mitochondrial dysfunctionABSTRACTCalpains, Ca2+-activated cysteine proteases, are cytosolic enzymes implicated in numerous cellular functions and pathologies. We identified a mitochondrial Ca2+-inducible protease that hydrolyzed a calpain substrate (SLLVY-AMC) and was inhibited by active sitedirected calpain inhibitors as calpain 10, an atypical calpain lacking domain IV. Immunoblot analysis and activity assays revealed calpain 10 in the mitochondrial outer membrane, intermembrane space, inner membrane, and matrix fractions. Mitochondrial staining was observed when COOH-terminal green fluorescent protein-tagged calpain 10 was overexpressed in NIH-3T3 cells and the mitochondrial targeting sequence was localized to the NH2-terminal 15 amino acids. Overexpression of mitochondrial calpain 10 resulted in mitochondrial swelling and autophagy that was blocked by the mitochondrial permeability transition (MPT) inhibitor cyclosporine A. With the use of isolated mitochondria, Ca2+-induced MPT was partially decreased by calpain inhibitors. More importantly, Ca2+-induced inhibition of Complex I of the electron transport chain was blocked by calpain inhibitors and two Complex I proteins were identified as targets of mitochondrial calpain 10, NDUFV2, and ND6. In conclusion, calpain 10 is the first reported mitochondrially targeted calpain and is a mediator of mitochondrial dysfunction through the cleavage of Complex I subunits and activation of MPT. protease; respiration 2+ CALPAINS, Ca -activated cysteine proteases, are involved in several physiological and disease processes: calpains are linked to the pathogenesis of Alzheimer's disease, cataracts, limb-girdle muscular dystrophy, gastric carcinoma, and Type II diabetes (30). Calpains cleave diverse protein species, including receptors (32), ion channels (13, 35), cytoskeletal components (44, 48, 54, 61), proteases (15,16, 23), oncogenic proteins (50), and cell signaling molecules (56, 63). Calpains also are thought to participate in the induction of cellular necrosis in various cell types (38, 59, 66, 71) and in the regulation of apoptosis through interactions with p53, Bid, and caspase 3 (15, 21, 50). Unfortunately, in many cases, the specific calpain responsible for these cellular actions and disease processes has not been identified. While calpains are a 14-member family, only a few calpain isoforms have been studied extensively. Calpains 1 and 2 are ubiquitously expressed cytosolic enzymes that dimerize with a small calpain subunit (calpain 4). Calpains 1 and 2 are 80 kDa and consist of four domains (I-IV). Domain I is an autolytic domain often cleaved during calpain activation. Domain II contains the catalytic active site where histidine, cysteine, and asparagine residues critical

for proteolysis are located. Domain III has a C2-like phospholipidbinding domain, and Domain IV contains a Ca2+-binding penta-EF-hand motif common to many Ca2+-binding proteins. Calpains are commonly separated into two groups, typical and atypical, based on the presence or absence of Domain IV (30). The absence of a typical domain IV may impart novel functions, substrate affinities, and activation/inhibition mechanisms to the more understudied atypical calpains (24). While calpains are generally thought to be cytosolic proteins, Hood et al. (36) has suggested that calpain 1, previously thought to be solely cytosolic, also localizes to Golgi and endoplasmic reticular membranes. Moreover, there are numerous reports of a mitochondrial calpain-like activity (1, 4, 9, 19, 31, 52, 60). However, cytosolic contamination of the mitochondrial preparations has been a concern, and investigators have inconsistently described the submitochondrial location of this calpain-like activity (matrix fraction vs. intermembrane space). At this time, a specific mitochondrial calpain has not been identified. The function of a putative mitochondrial calpain has not been elucidated. However, Ca2+sensitive proteolytic activities in mitochondria have been associated with the cleavage of RXR (19), preornithine transcarbamylase processing (47), cleavage of nuclear poly-ADP ribosepolymerase, and aspartate aminotransferase (26), and induction of MPT (31, 52). Furthermore, mitochondria are dynamic organelles participating in cellular Ca2+ regulation and are capable of buffering large amounts of cytosolic Ca2+ (20). Mitochondrial Ca2+ uptake is known to induce the mitochondrial permeability transition (MPT) and mitochondrial dysfunction (2, 65). In the present study, we have taken a molecular approach to identify the mitochondrial calpain and have evaluated its role in mitochondrial dysfunction.

MATERIALS AND METHODSMitochondrial isolation and fractionation. Kidney mitochondria were isolated from male Sprague-Dawley rats (250 g) and female New Zealand White rabbits (2 kg), as previously described (57, 69). Briefly, the kidney cortex was minced and homogenized in ice-cold buffer A (0.27 M sucrose, 5 mM TrisHCl, and 1 mM EGTA, pH 7.4). Nuclei and cellular debris were pelleted by centrifugation at 600 gfor 5 min. The supernatant was centrifuged at 7,700 g for 5 min, resulting in a crude mitochondrial pellet. The pellet was washed once in ice-cold 0.27 M sucrose and resuspended in buffer B (130 mM KCl, 9 mM Tris-PO4, 4 mM TrisHCl, and 1 mM EGTA, pH 7.4). Crude mitochondria were then layered onto a sucrose/Percoll gradient and centrifuged at 20,000 g for 20 min. Purified mitochondria were subfractionated as described previously (70). Outer membrane rupture was achieved by hypotonic lysis in icecold buffer C (10 mM KH2PO4, pH 7.4) for 20 min at 0C. Mitoplasts were separated from the supernatant by centrifugation at 7,700 g for 5 min. The outer membrane fraction was obtained by centrifugation of the supernatant at 54,000 g for 30 min. The resulting pellet was resuspended in ice-cold buffer D (300 mM sucrose, 1 mM EGTA, and 20 mM MOPS, pH 7.4) and sonicated five times in 30-s bursts. The inner membrane and matrix fractions were then separated by centrifugation at 54,000 g for 30 min. Outer and inner membrane fractions were resuspended inbuffer D. Fraction purity was assayed via marker

enzyme analysis (outer membrane, monoamine oxidase; intermembrane space, adenylate kinase; inner membrane, cytochrome oxidase; matrix, fumarase). The activities of monoamine oxidase (57a), adenylate kinase (12), cytochrome oxidase (49), and fumarase (72) were determined by standard methods. Fractions were frozen at 70C for subsequent immunoblot analysis. Calpain activity. Calpain activity was assayed spectrophotometrically using the calpain-specific substrate SLLVY-AMC (Bachem), as previously described (6). Whole, energized, mitochondria (200 g) were diluted in buffer B and incubated with various concentrations of CaCl2 in the presence of 50 M SLLVY-AMC. Activity was measured under linear conditions as a function of AMC hydrolysis using excitation and emission wavelengths of 355 and 444 nm, respectively. Mitochondria incubated in the absence of substrate exhibited the same fluorescence as buffer B alone. Respiratory complex activity. Complex I enzyme activity was measured as previously described (14). The assay medium was composed of antimycin A (2 g/ml), ubiquinone (65 M), NADH (130 M), and KCN (2 mM) in a phosphate buffer (25 mM potassium phosphate, 5 mM MgCl2, and 2.5 mg/ml BSA, pH 7.2). Mitochondria (2050 g) were then added and NADH oxidation was measured spectrophotometrically at 340 nm for 35 min before the addition of rotenone (2 g/ml). Absorbance changes were measured for another 3 min and Complex I activity reported as the rotenone-sensitive NADH:ubiquinone oxidoreductase activity. Complex II enzyme activity was measured as previously described (14). Briefly, mitochondria (2050 g) were preincubated in assay media (25 mM potassium phosphate and 5 mM MgCl2, pH 7.2) and 20 mM succinate for 10 min at 30C. Antimycin A (2 g/ml), 2 mM KCN, 2 g/ml rotenone, and 50 M 2,6-dichlorophenolindophenol were added, and absorbance at 600 nm recorded for 3 min. The reaction was then initiated with ubiquinone (65 M), and absorbance monitored for 35 min. Complex III activity was measured as previously described (14). Briefly, 15 M cytochrome c (III), 2 g/ml rotenone, 0.6 mM dodecyl-b-Dmaltoside, and 35 M ubiquinol-2 were added to assay media [25 mM potassium phosphate, 5 mM MgCl2, 2.5 mg/ml BSA (fraction V), 2 mM KCN, pH 7.2] and the nonenzymatic rate of reduction of cytochrome c measured for 1 min at 550 nm. To initiate the reaction, mitochondria (520 g) were added, and the initial increase in absorbance was measured for 2 min. Measurement of renal cortical mitochondria oxygen consumption. Oxygen consumption was monitored as previously described (57) using a six-chambered oxymeter and computer interface (model 928; Strathkelvin, Glasgow, UK). Renal cortical mitochondria (RCM) were suspended at 1.3 mg mitochondrial protein/ml in mitochondrial incubation buffer with pyruvate/malate (5/5 mM) as the respiratory substrates. In some experiments, succinate (10 mM) or ascorbic acid/tetramethylphenylene diamine (TMPD; 5/0.5 mM) served as the respiratory substrates in the presence of 100 M

rotenone (a Complex I inhibitor) or 2.5 M antimycin A (a Complex III inhibitor), respectively. Mitochondria were gassed (5% CO2-95% O2) for 5 min before treatments and measurement of respiration. The respiration chamber was maintained at 37C and stirred magnetically. After the basal rate (state 4) of O2 consumption was determined, ADP (final concentration = 1 mM) was injected to obtain state 3 respiration. Only mitochondria with respiratory control ratios (RCRs: state 3/state 4) 4 were used for experiments to ensure that test mitochondria were tightly coupled. In some experiments, respiration measurements were performed in the presence or absence of 1 M Ca2+ over various time courses. For inhibition experiments, calpain inhibitors were added 30 min before the addition of 1 M Ca2+ and respiration measured after 5 min. Ca2+ was added to buffer B in all experiments such that Ca2+ concentrations were maintained at 1 M. Mitochondrial swelling. RCM swelling was assessed spectrophotometrically as previously described (1). Briefly, RCM were suspended at a final concentration of 1 mg/ml of mitochondrial protein in buffer B supplemented with pyruvate/malate (5/5 mM) and absorbance measured for 10 min at 540 nM. After basal measurements were taken, Ca2+ was added (1 M final sustained Ca2+concentration) and absorbance was monitored for an additional 5 min and swelling rates (A/min) determined. Zymography. Zymography was performed as previously described (5) with minor modifications. Zymogram gels were cast immediately before electrophoresis and consisted of a 10% nondenaturing acrylamide resolving gel and an 8% stacking gel. Resolving gels were copolymerized with the calpain substrates FITC-casein (10 mg/ml) or SLLVY-AMC (50 M). Protein samples (200 g) and purified porcine calpains 1 and 2 (2 g) (Calbiochem) were loaded and subjected to electrophoresis in a nondenaturing running buffer (125 mM Tris base, 625 mM glycine, and 5 mM EGTA, pH 8.0) at 120 V for 2 h at 4C. Gels were subsequently bathed in Ca2+incubation buffer (50 mM TrisHCl, 5 mM CaCl2, and 10 mM 2-mercaptoethanol, pH 7.0) overnight at 4C and imaged on an Alpha Innotech imaging station fitted with a FITC filter. Immunoblot analysis. Isolated mitochondrial fractions were subjected to SDS-PAGE (412% acrylamide) and transferred to nitrocellulose membranes. Membranes were incubated with primary antibodies to mcalpain, -calpain, calpain 10, NDUFV2, DAP13, and ND6. The primary antibodies used were monoclonal rabbit anti-human m-calpain (1:1,000; Affinity Bioreagents), rabbit anti-human m-calpain (domain IV) (1:1,000; Calbiochem), rabbit anti-human -calpain (domain III-IV) (1:1,000; Abcam), 1:200 rabbit anti-rat calpain 10 (domain II; generously provided by Tom Shearer, Oregon Health and Science University, Portland, OR), rabbit anti-human calpain 10 (1:1,000; domain III, Abcam), rabbit anti-rat calpain 10 (1:1,000; domain II, Biogenesis), rabbit anti-NDUFV2 (1:200; generously provided by Dr. Yamaguchi, The Scripps Research Institute, La Jolla, CA), monoclonal mouse anti-human DAP13 (1 g/ml; Genway Biotech), and monoclonal

mouse anti-human ND6 (1 g/ml; Molecular Probes). Antibody incubation was followed by a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:1,000; Santa Cruz). Immunoreactive protein was visualized by enhanced chemiluminesence (Amersham) and imaged using an Alpha Innotech imaging station. Plasmid construction. cDNA for human calpain 10a (BC004260 [GenBank] ) was obtained from ATCC in the pOTB7 shuttle vector. Full-length calpain 10 was amplified by PCR (sense: 5'TGGGAGCCCGCGGAGCCGAG-3'; antisense: 5'TCATCACTGCCATGACGGAGACCTC-3') and subcloned into pcDNA3.1TOPO-TA-CT-green fluorescent protein (GFP) (pcDNA3.1-CAPN10-GFP) (Invitrogen) producing a calpain 10-GFP fusion product (COOH terminal GFP). GFP control plasmids coding for cytosolic GFP and mitochondrially targeted GFP (cytochrome oxidase IV signal sequence) were generous gifts from Dr. Douglas Sweet (Medical University of South Carolina, Charleston, SC). Complementary DNA sequences coding for the N-terminal 15 amino acids of calpain 10 were generated, annealed, and ligated into pcDNA3.1-TOPO-TA-CT-GFP (pcDNA3.1-TS-GFP) to assess NH2-terminal sufficiency for mitochondrial targeting. The negative control for this experiment was obtained via ligation of the above sequence into pcDNA3.1- TOPO-TA-CT-GFP (pcDNA3.1-TSINV-GFP) in the reverse orientation. Cell culture and transfection. NIH-3T3 fibroblasts were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum until confluent. Cells were split and plated onto 35-mm confocal dishes (MatTek) at a density of 250,000 cells/plate. At 70% confluence, cells were transiently transfected with 1 g pcDNA3.1-CAPN10-GFP, pcDNA3.1-TS-GFP, or pcDNA3.1-TSINV-GFP plasmid constructs using Lipofectamine 2000 (Invitrogen). Selected plates were treated with 6 M cyclosporine A, 5 mM 3-methyladenine, or vehicle (DMSO) 4 h after transfection. Cells were incubated for 24 h, and, when indicated, exposed to 50 nM MitoTracker Red (Molecular Probes) and/or 100 nM LysoTracker Red (Molecular Probes) for 20 min before confocal microscopy imaging. Cells were imaged using a Zeiss LSM 5 confocal microscope using multiple tracks to eliminate fluorescent cross-talk. Statistical analysis. RCM isolated from one rabbit represent one experiment (n = 1). The appropriate ANOVA was performed for each data set by using SigmaStat statistical software. Individual means were compared with Fisher's protected least-significant difference test, with P 0.05 being considered a statistically significant difference between mean values. Means with different lettered subscripts within groups are significantly different from each other, P 0.05. Linear regression was also performed by using SigmaStat statistical software.

RESULTSCalpain activity in isolated mitochondria and submitochondrial fractions. To determine the presence of calpain activity, rabbit RCM were

incubated with increasing concentrations of Ca2+ in the presence of the calpain substrate SLLVY-AMC. RCM cleavage of SLLVY-AMC occurred in the absence

of added Ca2+, was linear over time (data not shown), and increased in the presence of increasing Ca2+ concentrations (Fig. 1A) to a maximum of 120% of control at 10 M Ca2+. The addition of increasing concentrations of the Ca2+ chelator EGTA to RCM permeabilized with 0.5% digitonin, in the presence of 1 M Ca2+, produced a maximal 50% decrease in SLLVY-AMC hydrolysis (Fig. 1B). Blockade of the Ca2+uniporter with ruthenium red also inhibited Ca2+induced hydrolysis of SLLVY-AMC in RCM (Fig. 1C). Ca2+-activated SLLCYAMC hydrolysis was inhibited in a concentration-dependent manner by the calpain inhibitors calpeptin and E-64, but not by the calpain inhibitor PD150606 (Fig. 1D). These data reveal that RCM exhibit basal calpain activity, that exogenous Ca2+ increases calpain activity in a ruthenium red-sensitive manner, that calpain activity exists in the absence of Ca2+, and that the calpain inhibitors calpeptin and E-64, but not PD150606, blocked the calpain activity. Because ruthenium red blocks Ca2+ uptake across the mitochondrial inner membrane and the increase in calpain activity produced by exogenous Ca2+ was ruthenium red sensitive, we suggest that the calpain activity is located in the mitochondrial matrix. Because the calpain activity was insensitive to PD150606, which binds to domain IV of typical calpains (67), we suggest that the RCM calpain lacks the penta-EF-hand domain and is a member of the atypical calpain family. .To determine which submitochondrial fraction(s) contain calpain activity, RCM were fractionated into outer membrane, intermembrane space, inner membrane, and matrix fractions. Marker enzymes (monoamine oxidase, outer membrane; adenylate cyclase, intermembrane space; cytochrome oxidase, inner membrane; fumarase, matrix) were used to assess mitochondrial fraction purity (Table 1). Ca2+-activated hydrolysis of SLLVY-AMC was observed in each fraction with the outer membrane having the highest activity and the matrix the least on a per mg protein basis. Maximal activity was observed at 2 mM Ca2+ in the outer membrane and at 10 mM for the remaining three fractions (Fig. 1E). These results reveal that calpain activity is present in multiple mitochondrial fractions. FITC-casein zymography is a technique commonly used to evaluate calpain activity in cell lysates. We employed this technique to evaluate calpain activity in rabbit RCM fractions and in the cytosol from renal cortical cells. Equal amounts of total protein (200 g) from cytosolic, mitochondrial, and submitochondrial fractions were loaded into individual zymogram gels. Two bands of activity were observed in the cytosol corresponding to calpains 1 and 2 (Fig. 2A), both known to exist in the cytosol of renal proximal tubule cells (44). However, no bands were detected in the sample lanes for whole mitochondria or mitochondrial subfractions, suggesting that the mitochondria harbored no endogenous calpain 1 or 2, that the mitochondrial preparation was free of cytosolic contamination, and that mitochondrial calpain exhibits a substrate specificity different from that of the typical calpains. We modified this assay to evaluate mitochondrial calpain activity by replacing FITC-casein with SLLVY-AMC (a known mitochondrial calpain substrate in vitro) as the calpain substrate. AMC is liberated by

proteolytic activity to generate fluorescent bands in the gel. Purified calpains 1 and 2 were used as controls and fluorescent bands were identified in the presence of Ca2+(Fig. 2B). No fluorescent bands were observed in the outer membrane and intermembrane space fractions in the presence of Ca2+. In contrast, one strongly fluorescent band and two weaker bands were observed in the matrix fraction. These results reveal three distinct calpain activities in the matrix fraction that are Ca2+-inducible and are different than calpains 1 or 2. No activity was observed in the outer membrane, inner membrane, or intermembrane space fractions. Identification of calpain 10 in mitochondrial subfractions. The protein localization algorithm MITOP2 (http://ihg.gsf.de/mitop2/start.jsp) was used to determine which of the 15 calpain isoforms would be predicted to localize to mitochondria. While a negative score using this algorithm does not exclude the possibility of mitochondrial localization, this algorithm only returned a positive score for calpain 10. Cytosolic and RCM fractions from rabbit and rat kidney cortex were probed for calpain 10 with three separate antibodies directed against different calpain 10 domains using immunoblot analysis. The presence of calpains 1 and 2 also were examined. A 75 kDa band was identified in all rabbit and rat RCM fractions using all three calpain 10 antibodies, albeit at different levels (Fig. 3A). An immunoreactive band also was identified in the cytosol using an antibody against calpain 10. Immunoblots probed with antibodies against calpains 1 and 2 revealed immunoreactive proteins against their respective purified proteins and cytosol but not in any of the RCM fractions (Fig. 3A). These results reveal that calpain 10 also resides in the mitochondria in at least two species (rabbit and rat), and is present in all RCM fractions. Furthermore, the bands of immunoreactivity are consistent with the largest calpain 10a isoform (75 kDa) (37), indicating the absence of import signal cleavage. The strong fluorescent band identified in the matrix sample following zymography was eluted and subjected to SDS/PAGE and immunoblot analysis using an anti-calpain 10 antibody (Fig. 3B). These results reveal that the mitochondrial matrix calpain activity observed during zymography is calpain 10. Calpain 10 is targeted to mitochondria via an NH2 terminal targeting peptide.Human calpain 10a was subcloned into a TOPO-TA-CT-GFP vector (Invitrogen) to produce a calpain 10-GFP fusion protein containing an intact NH2-terminus and a COOH-terminal GFP moiety. This construct was chosen to avoid modification of the NH2 terminus, which could result in the loss of putative mitochondrial localization motifs. NIH-3T3 cells were transfected with the pcDNA3.1-CAPN10-GFP construct, exposed to MitoTracker and/or LysoTracker Red, and imaged via confocal microscopy. Cells also were transfected using GFP constructs specific for cytosol (data not shown) or mitochondria (Fig. 4A) to serve as positive GFP controls for these compartments.

During initial experiments, we observed two distinct populations of cells after transfection with the CAPN10-GFP construct. All transfected cells demonstrated GFP expression that was localized to intracellular compartments with some being punctuate in nature and others exhibiting larger globular patterns of GFP expression. The latter of these two groups of cells exhibited altered cellular morphology, decreased plate adhesion, and the formation of autophagocytic vesicles as shown by costaining with LysoTracker Red (Fig. 4B). The former of these two groups of cells exhibited normal cellular morphology but contained swollen mitochondria (Fig. 4C) compared with mitochondrially targeted GFP controls (Fig. 4A). These data conclusively show the targeting of calpain 10 to the mitochondria and suggest that calpain 10 may play a role in mitochondrial dysfunction and/or swelling as evidenced by the changes seen in mitochondrial morphology.

To test the hypothesis that the NH2 terminus of calpain 10 is responsible for mitochondrial localization, oligonucleotides coding for the first 15 NH2-terminal amino acid residues (Fig. 5B) were annealed and ligated into the TOPO-TA-CTGFP vector. Negative controls included the same oligonucleotides inserted in the reverse orientation. NIH-3T3 cells transfected with these constructs displayed cytosolic targeting and mitochondrial targeting for the reverse and forward orientations, respectively (Fig. 5A). These results reveal that the NH2-terminal 15 amino acids of calpain 10 are sufficient for mitochondrial targeting. We also analyzed the NH2 terminus of calpain 10 using LaTeX software package TeXtopo designed to display peptide sequences in an alpha helix representation (Fig. 5B). The results revealed that the NH2-terminus of calpain 10 can form the classic mitochondrial targeting motif, the amphipathic helix. three independent experiments. B: this helical wheel diagram shows a representation of the calpain 10 NH2terminus as if it were a helix using the TexTopo LaTeX package. The putative helix is amphipathic in nature, containing hydrophobic and aliphatic residues on one half and positively charged residues on the other. Adjacent to the helical wheel diagram is a schematic representation of calpain 10 and its NH2-terminal 15 amino acids used to engineer the calpain 10 targeting sequence-GFP construct (asterisks denote positively charged residues). The white bars in A represent a distance of 10 m.Mitochondrial swelling induced by overexpression of calpain 10 is sensitive to cyclosporine A and 3-methyladenine. As discussed above, overexpression of calpain 10 resulted in the conversion of the normal branched reticular mitochondrial morphology of control cells (Fig. 4A) to that of smaller, round and swollen organelles (Fig. 4C). Thus we hypothesized that the calpain 10-induced mitochondrial swelling may be sensitive to cyclosporine A [an inhibitor of MPT (62)] or 3methyladenine [an inhibitor of MPT and autophagocytosis (62)]. To test this hypothesis, cells were transfected with the calpain 10-GFP construct, treated with cyclosporine A or 3-methyladenine, and the

mitochondria was examined by confocal microscopy. Cyclosporine A and 3-methyladenine preserved the normal branched mitochondrial morphology and integrity (Fig. 4, D and E, respectively) compared with cells expressing calpain 10-GFP in the absence of MPT inhibitors, suggesting that calpain 10 may mediate mitochondrial swelling, in part, through the formation of the MPT pore. However, MPT formation was not explicitly examined in these experiments and it is possible that the mitochondrial fragmentation observed in our studies could also result from mitochondrial depolarization, electron transport chain dysfunction, or changes in mitochondrial fission or fusion proteins. To further examine the possible role of calpain 10 in MPT, RCM were treated with 1 M Ca2+ in the presence and absence of cyclosporine A or calpeptin, and RCM swelling determined. The addition of Ca2+induced RCM swelling, which was blocked by cyclosporine A (Fig. 6A). Mitochondrial pretreatment with calpeptin blocked 30% of Ca2+induced RCM swelling, indicating a role for calpain 10 in the formation of the MPT pore (Fig. 6B). . Calpain 10 mediates Ca2+-induced mitochondrial dysfunction. As shown in Fig. 4B, calpain 10 overexpression resulted in a fragmented mitochondrial morphology. This type of organellar derangement is seen not only during MPT, but is also characteristic of mitochondria with damaged or impaired components of the electron transport chain (ETC) (41). Thus calpain 10 may mediate ETC dysfunction. To test this hypothesis, RCM were isolated and suspended in incubation buffer containing the model Complex I substrates pyruvate/malate (5/5 mM). Mitochondrial oxygen consumption was then measured over time after treatment with 1 M Ca2+ in the presence of 1 mM ADP. State 3 respiration was unchanged under normoxic and hypoxic conditions in the absence of Ca2+ (Fig. 7A), and all future respiratory measurements were performed under normoxic conditions. The addition of 1 M Ca2+ produced a time-dependent decrease in mitochondrial state 3 respiration (Fig. 7B), and all subsequent experiments were conducted following the incubation of RCM with 1 M Ca2+ for 5 min. Experiments including inhibitors were performed after a 30 min preincubation period with specific calpain inhibitors. Pretreatment of RCM with increasing concentrations of the active-site calpain inhibitors calpeptin and E-64, but not the domain IV calpain inhibitor PD150606 preserved mitochondrial state 3 respiration (Fig. 7C). Higher concentrations of E64 did not produce enhanced state 3 respiration, most likely due to its poor membrane permeability. Simultaneous measurement of state 3 dysfunction and mitochondrial calpain activity demonstrated a strong correlation between these two end points (Fig. 7D). To determine the site of ETC dysfunction, isolated RCM were suspended in incubation buffer containing pyruvate/malate (Complex I substrates), succinate/rotenone (Complex II substrate/Complex I inhibitor), or ascorbate-TMPD/antimycin A (Complex IV electron donor/Complex III inhibitor). Mitochondrial state 3 respiration was then measured after treatment with 1 M Ca2+ for 5 min. Respiratory deficits

were observed only in the treatment group containing the Complex I substrates (Fig. 8A). To verify these findings, we performed specific Complex I, II, and III activity assays and determined that only Complex I activity was significantly decreased after Ca2+ exposure (Fig. 8B) and that this activity could be preserved by pretreatment with calpeptin. These findings show that Ca2+-induced respiratory dysfunction is limited to Complex I under these conditions and that electron transport in Complexes II-IV remains intact. Calpain 10 cleaves mitochondrial complex I subunits. Complex I of the ETC is composed of 46 individual subunits. We sought to determine the complex I protein substrate(s) of calpain 10. To do this, we analyzed each of the 46 Complex I subunits for possible calpain cleavage motifs using the PEST-FIND algorithm (http://srs.nchc.org.tw/embossbin/emboss.pl?_action=input&_app=pestfind). The PEST-FIND algorithm is useful for the identification of possible calpain and proteasome substrates, but does not predict actual cleavage sites within protein targets. Subsequent to PEST analysis, four protein species were identified as potential calpain substrates; NDUFV2, DAP13, NDUFS7, and ND6. Antibodies were available and obtained for NDUFV2, DAP13, and ND6. RCM were isolated and pretreated with various inhibitors for 30 min before the addition of Ca2+. RCM were treated with 1 M Ca2+ for 5 min, pelleted by centrifugation, and resuspended in buffer deficient in Ca2+ and containing a protease inhibitor cocktail (Sigma) to stop all proteolytic reactions. Aliquots were then subjected to immunoblot analysis using antibodies against NDUFV2, DAP13 and the ND6 subunits of Complex I. The calpain inhibitors ALLN and calpeptin completely inhibited NDUFV2 and ND6 hydrolysis while pretreatment with the serine protease inhibitor bestatin did not (Fig. 8C). DAP13 did not undergo hydrolysis (Fig. 8C). Pretreatment with ruthenium red also blocked NDUFV2 and ND6 degradation (Fig. 8C), suggesting that calpain 10-mediated proteolysis of Complex I subunits occurs in the matrix. Our results demonstrate Ca2+-induced calpain-dependent proteolysis of the Complex I subunits NDUFV2 and ND6, but not of the DAP13 subunit.

DISCUSSIONCalpains are ubiquitously expressed throughout eukaryotic organisms and are involved in numerous cellular functions (30). Within mammals, calpains 1, 2, 4, 5, 7, 10, 12, and 13 are ubiquitously expressed, whereas calpains 3, 6, 8, 9, and 11 have more tissue-specific distributions (37), implicating a diverse set of roles for calpain family members. However, the physiological and pathological roles of most calpain isoforms have not been examined. While calpains are thought to be cytosolic proteins, investigators have reported calpain-like activities in isolated mitochondria (1, 4, 9, 19, 31, 52, 60). Using multiple approaches, we identified a resident mitochondrial calpain as calpain 10 and determined that it plays a role in mitochondrial dysfunction.

Ma et al. (45) first cloned calpain 10 in 2001 and proposed that this calpain isoform was localized to the cytosol and translocated to the nucleus after increases in cellular Ca2+. Our data reveal that calpain 10 is present in the cytosol and mitochondria, while significant nuclear staining is absent under nonstimulated conditions (Fig. 4C). However, in the present study, we have not evaluated the effects of Ca2+ overload on calpain 10 subcellular localization. We believe that our cloning strategy avoids the complications and cross-reactivity of antibodies used for immunohistochemistry and provides a clearer picture of the subcellular localization of calpain 10. Many mitochondrial matrix-targeted proteins contain either a NH2- or COOH-terminal signaling motif (25, 58). This motif most commonly takes the form of an NH2-terminal amphipathic helix containing positively charged residues on one half of the helix and hydrophobic residues on the other (58). We determined the presence of such a motif in calpain 10 using LaTeX software package TeXtopo (Fig. 5B), designed to display peptide sequences in a helical representation (10). We tested the hypothesis that the NH2-terminal 15 amino acids of calpain 10 are responsible for mitochondrial localization, and found that cells expressing GFP conjugated to the NH2-terminal 15 amino acids localized to the mitochondria whereas cells expressing GFP conjugated to the same nucleotides inserted in the reverse orientation remained in the cytosol. Investigators have previously reported a Ca2+-inducible calpain-like activity in mitochondria (1, 9, 52, 60). Our data reveal that calpain activity is present in intact mitochondria and is increased following Ca2+ addition. Because Ca2+ is concentrated in the mitochondrial matrix, and ruthenium red blocked the Ca2+-induced increase in calpain activity, we propose that the majority of Ca2+-inducible calpain 10 activity in mitochondria is localized to the matrix. This idea is supported by the experiment in which calpain 10 was identified in the matrix fraction following zymography. Using permeabilized mitochondria and Ca2+chelation, we observed that 50% of the mitochondrial calpain 10 activity was Ca2+ dependent. It is unclear whether the remaining cysteine protease activity in the matrix was due to Ca2+independent calpain 10 activity or due to the activity of another cysteine protease. One tempting explanation is that some of the Ca2+independent activity seen in isolated RCM subfractions may come from one of the remaining 7 calpain 10 splice variants (all of which carry the mitochondrial targeting signal). It is also possible that calpain 10 is less Ca2+ dependent than the typical calpains due to its lack of a Domain IV or that it is dually regulated by both calcium and some sort of secondary protein modification such as phosphorylation (an event shown to activate calpain 2) (29). More research is necessary to evaluate the relationship between Ca2+ and calpain 10 using purified protein. Interestingly, a recent report from Garcia et al. (28), has proposed that calpain 1 is localized to both the cytosol and mitochondrial fractions of rat brain cortex and of SH-SY5Y human neuroblastoma cells. Our data are not consistent with the mitochondrial localization of calpain 1 in

kidney mitochondria. For example, we assayed for calpain 1 in purified mitochondria and cytosol by both immunoblot and FITC-casein zymography. While calpain 1 was identified in the cytosol it was not present in mitochondria. Furthermore, calpain 1 does not have an identifiable mitochondrial targeting signal as determined by the MITOP2 algorithm, although this does not completely exclude the possibility of mitochondrial localization. Finally, the "typical" calpain inhibitor PD150606 (a membrane permeable inhibitor) blocks calpain 1 but did not block calpain activity in mitochondria. Excluding model differences, one possible explanation may be that calpain 1 can associate with the mitochondrial outer membrane as described for the Golgi and endoplasmic reticulum (36), but is incapable of penetrating into the mitochondrial interior. Using isolated RCM fractions, we observed calpain activity and calpain 10 protein in the outer membrane, intermembrane space, inner membrane, and matrix fractions. These results support the work of others who have reported calpain activity in more than one mitochondrial fraction (9). However, using zymography, Ca2+-inducible calpain 10 activity was primarily observed in the matrix fraction and increases in calpain activity following Ca2+ addition were ruthenium red-sensitive, suggesting matrix calpain 10 activity. While the reasons for these differences are not known, we propose that Ca2+-inducible calpain 10 activity in intact mitochondria is primarily the result of matrix calpain 10 and that calpain 10 may be inactive in the remaining fractions or regulated in a Ca2+-independent manner. Ca2+ activation of calpains is important for physiological functions, but excessive Ca2+ can produce abnormal proteolytic activity and cell injury and death (44) (See Fig. 9). MPT is a form of mitochondrial dysfunction produced by Ca2+ overload, decreased adenine nucleotide concentrations, decreased mitochondrial membrane potential, and increased oxidative stress, and is characterized by the opening of a pore and mitochondrial swelling (2, 33, 43). Calpain 10 overexpression induced mitochondrial fragmentation and swelling, consistent with MPT (50) and this altered mitochondrial morphology was blocked by two MPT inhibitors. In addition, high levels of calpain 10 expression induced mitochondrial autophagy, a process blocked by 3-methyladenine and thought to be stimulated by MPT induction (42, 43, 55). We demonstrated 30% inhibition of MPT by the calpain inhibitor calpeptin, which agrees with Gores et al. (1, 31), who reported a liver mitochondrial calpain-like activity that regulated MPT. However, 70% of Ca2+-induced MPT was not calpain dependent. It is conceivable that calpain 10 does not directly regulate MPT per se, and the mitochondrial fragmentation seen in our studies may be regulated via other calpain 10-mediated proteolytic events. Nevertheless, cyclosporine A and 3methyladenine blocked the mitochondrial fragmentation and swelling observed subsequent to calpain 10 overexpression. Thus, the observation that cyclosporine A and 3-methyladenine did not block calpain 10-GFP staining, but preserved mitochondrial morphology supports our thoughts.

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Fig. 9. Proposed mechanism of action for calpain 10-mediated ETC dysfunction. Depicted above is a schematic diagram of the proposed mechanism of calpain 10 action after Ca2+-induced mitochondrial dysfunction. After injury, increases in cytosolic Ca2+ are translated to the mitochondria via the ruthenium red-sensitive calcium channel uniporter. Increases in matrix Ca2+ activate mitochondrial calpain 10, inducing cleavage of two critical subunits of Complex I, ND6 (*) and NDUFV2 (#), leading to subsequent respiratory deficits. Increases in mitochondrial calcium also contribute to the induction of MPT, possibly through proteolysis of membrane pore proteins. The mitochondrial ETC provides a mechanism for the generation of ATP through the oxidative phosphorylation of ADP. As previously reported (65), mitochondrial Ca2+ overload results in decreased ETC activity. Our results reveal that Complex I is the most sensitive complex to Ca2+induced mitochondrial dysfunction and that calpain inhibition with calpeptin protects against Complex I dysfunction. We sought to determine potential Complex I protein substrate(s) of calpain 10 using the PEST-FIND algorithm (http://srs.nchc.org). Four Complex I proteins were identified and three proteins were tested (NDUFV2, DAP13, and ND6). Ca2+-induced inhibition of ETC was associated with hydrolysis of NDUFV2 and ND6 but not DAP13. Further, pretreatment with ruthenium red also blocked NDUFV2 and ND6 degradation, suggesting that Ca2+-induced calpain 10-mediated proteolysis of NDUFV2 and ND6 subunits occurs in the mitochondrial matrix. Deficiencies or mutations in various protein subunits of Complexes I-IV have been identified and linked to clinical syndromes (27). Complex I subunit mutations are perhaps the most common and account for 33% of all respiratory chain disorders (64). In addition, Ricci et al. (53) have shown that the proteolysis of Complex I subunits can also contribute to ETC dysfunction by demonstrating the caspase-mediated cleavage of the 75 kDa NDUFS1 subunit. In the current study, we have identified two Complex I subunits, NDUFV2 and ND6, which undergo calpainmediated hydrolysis. The NDUFV2 subunit is a nuclear-encoded 24-kDa protein found in the matrix arm of Complex I and is required for Complex I activity (3, 40). Defects in this protein have been identified previously and result in encephalopathies and bipolar disorder (11, 68). The ND6 subunit, a 20-kDa protein encoded by the mitochondrial genome, is a transmembrane protein known to assist in Complex I assembly, is required for Complex I activity, and whose mutations are associated with Leber's hereditary optic neuropathy (7, 8, 17, 22).

Thus, similar to the mitochondrial calpain-induced hydrolysis of ND6 and NDUFV2 and the associated Complex I dysfunction, genetic alterations in these proteins result in inhibition of Complex I. Interestingly, Koopman et al. (41), have shown that single subunit mutations in Complex I not only reduce enzyme activity of the complex but are often associated with alterations in mitochondrial morphology; a phenomenon we observed with calpain 10 overexpression. In summary, we have identified the endogenous mitochondrial calpain as calpain 10 and that it plays a role in mitochondrial viability. While the physiological and pathological functions of calpain 10 have not been studied extensively, it has been linked to ryanodine-induced apoptosis, GLUT4 vesicle translocation, pancreatic -cell exocytosis, cataractogenesis, hypertriglyceridemia, and is genetically linked to Type II diabetes, a disease associated with mitochondrial dysfunction (18, 34, 39, 45, 46, 51). Mitochondria are increasingly being thought of as cell death checkpoints at which signals for necrosis and apoptosis are sent for downstream processing. It will be exciting to elucidate the role of calpain 10 in orchestrating these events and how we may be able to manipulate this system for therapeutic intervention in disease states dominated by the dysregulation of cellular Ca2+ homeostasis. .

Regulating cell migration: calpains make the cutSummaryThe calpain family of proteases has been implicated in cellular processes such as apoptosis, proliferation and cell migration. Calpains are involved in several key aspects of migration, including: adhesion and spreading; detachment of the rear; integrin- and growth-factor-mediated signaling; and membrane protrusion. Our understanding of how calpains are activated and regulated during cell migration has increased as studies have identified roles for calcium and phospholipid binding, autolysis, phosphorylation and inhibition by calpastatin in the modulation of calpain activity. Knockout and knockdown approaches have also contributed significantly to our knowledge of calpain biology, particularly with respect to the specific functions of different calpain isoforms. The mechanisms by which calpain-mediated proteolysis of individual substrates contributes to cell motility have begun to be addressed, and these efforts have revealed roles for proteolysis of specific substrates in integrin activation, adhesion complex turnover and membrane protrusion dynamics. Understanding these mechanisms should provide avenues for novel therapeutic strategies to treat pathological processes such as tumor metastasis and chronic inflammatory disease.

Key words Calpain Cell motility Proteolysis

IntroductionThe importance of cell migration is evident from both the physiological processes that depend on the regulated movement of cells, including embryonic development, immune responses and tissue maintenance and repair, and from the disease states driven by aberrant cell motility, such as chronic inflammation, vascular disease and tumor metastasis. Not surprisingly, immense effort has been directed towards furthering our understanding of this complex process. These efforts have provided us with the current concept of cell migration, which comprises a cycle of several highly coordinated and regulated steps (Lauffenburger and Horwitz, 1996;Ridley et al., 2003). In response to various migratory cues, directional movement is initiated by polarization of the cell, as defined by the spatial segregation of molecular

machineries that control the different stages of the migratory cycle. At the front of the cell, actin polymerization drives membrane protrusion to form the leading edge. Subsequently, the leading edge is stabilized by attachment to the extracellular matrix (ECM) through integrin-mediated adhesion complexes, which not only link the ECM to the actin cytoskeleton but also function as signal transduction centers that modulate cell migration. Once coupled to adhesion complexes, the actin cytoskeleton can generate the forces necessary to translocate the cell body forward. Finally, adhesive contacts at the rear of the cell must be disassembled to allow detachment of the rear and to complete the migratory cycle. Because of their involvement in cell motility, integrin-containing adhesion complexes are necessarily dynamic structures that undergo repeated cycles of formation and disassembly (Webb et al., 2002). Likewise, the activities of the actin-based protrusion and contraction machineries must also be continually regulated to ensure proper timing and localization (Rafelski and Theriot, 2004). The calpain family of proteases has been shown to contribute to the control of cell migration through their ability to regulate the dynamics of both integrin-mediated adhesion and actin-based membrane protrusion (Perrin and Huttenlocher, 2002). Although our current understanding of the mechanisms underlying this regulation remains limited, recent studies have begun to shed light on this subject. Here, we discuss recent advances that have provided insight into where calpains fit into the cell migration cycle, how the activities of calpains are modulated, the roles of individual calpain isoforms in motility, and the molecular basis of their effects during directional cell movement.

Calpain family of proteasesThe mammalian calpain gene superfamily contains 16 known genes: 14 of these genes encode proteins that contain cysteine protease domains; the other two genes encode smaller regulatory proteins that associate with some of the catalytic subunits to form heterodimeric proteases (Goll et al., 2003; Suzuki et al., 2004). Several calpain isoforms are ubiquitously expressed, whereas many demonstrate tissuespecific expression patterns (Table 1). They are typically thought of as intracellular proteases, although there is some evidence that active calpains are also found in the extracellular space (Nishihara et al., 2001;Xu and Deng, 2004). However, the physiological significance of extracellular calpains is not yet known. Within the cell, the localization patterns of calpains are complex and somewhat variable (Table 2), which means that their subcellular localization might be dynamically regulated and constitutes an important factor in the modulation of their functions.

Calpain structureThe enzymatically active (large) calpains each comprise up to four domains (Fig. 1) (Hosfield et al., 1999; Pal et al., 2003; Sorimachi and Suzuki, 2001;Strobl et al., 2000). Domain I is a single -helix present at the N-terminus of some calpains; it can interact with domain VI of the non-catalytic (small) subunits and may be important for stability. Domain II comprises the protease domain, which contains the active site catalytic triad Cys105, His262 and Asn286. Interestingly, the alignment and spacing of these residues in the inactive molecule is such that catalytic activity is not permitted, indicating that a structural change must take place to activate calpains. Domain III consists of eight -strands arranged in a -sandwich configuration similar to that of C2 domains. The C2 domain was first discovered in protein kinase C as a stretch of approximately 130 amino acids that binds phospholipids in a calciumdependent manner (Newton and Johnson, 1998). Since then, C2-like domains have been identified in nearly 100 proteins, and they are usually involved in binding calcium and phospholipids (Rizo and Sudhof, 1998). Domain III can bind phospholipids in a calcium-dependent fashion (Tompa et al., 2001), which further suggests that it is a C2-like domain. Domains IV and VI in the large and small

subunits, respectively, each contain five EF-hand motifs, the fifth EF hand from each subunit interacting with the other to assemble heterodimers (Blanchard et al., 1997; Hosfield et al., 1999; Lin et al., 1997). Domain V of the small subunits appears to have a very flexible structure as a consequence of being glycine rich, which is probably why, unlike the other domains, it remains unresolved by crystallography.

Calpain regulationCalpain activity is highly regulated in vivo by multiple mechanisms (Fig. 2A), although the details are only now beginning to be defined. The best-studied mechanism is activation by calcium - hence the name calpain (Guroff, 1964). In fact, calpain 1 and calpain 2 are commonly referred to by their in vitro requirements for calcium: calpain 1 (-calpain) is activated by micromolar calcium concentrations and calpain 2 (mcalpain) requires millimolar concentrations (Suzuki et al., 1981b). Because calpains contain calcium-binding EF-hand motifs in domains IV and VI and, because domain IV of calpain 1 and domain IV of calpain 2 are different, these were originally presumed to be responsible for the calcium-dependent activation of calpains. However, structural data suggest that conformational changes caused by calcium occupancy of the EF hands alone are insufficient to align the active site catalytic residues properly. Furthermore, functional studies have demonstrated that domain II alone exhibits calcium-dependent protease activity (Hata et al., 2001) and that non-EF-hand calcium-binding sites within the protease domain act as a calcium switch to align the catalytic triad (Hata et al., 2001; Moldoveanu et al., 2002; Moldoveanu et al., 2004).

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Fig. 1.

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Schematic representation of the domain architecture of the classical calpains. The 80 kDa large subunits can be divided into four domains, plus a short linker that might be important for transducing conformational changes throughout the molecule upon calcium binding (T). The N-terminal -helix makes up domain I, which interacts with the small subunits before undergoing intermolecular autolysis on activation. Protease activity is contained within domain II, which is further divided into subdomains (IIa and IIb) that make up the two halves of the active site. Domain III comprises a C2-like domain that harbors sites for phosphorylation and phospholipid binding. Five consecutive EF-hand motifs make up domain IV and contribute to the calcium binding of the large subunits and to dimerization with the small subunits. Domain VI of the small subunits has a similar arrangement; the first four EF hands participate in calcium binding and the last motif interacts with the large subunit. The small subunits also contain a highly flexible, glycine-rich region called domain V. Calpain 1 and calpain 2 large subunits are phosphorylated at several sites in domains I-III; some of these residues are conserved and some are isoform specific.


Fig. 2.


(A) Some of the mechanisms involved in regulating calpain activity. (B) Possible pathway for growth-factor-induced, calpain-mediated cell migration. Binding of epidermal growth factor (EGF) to its receptor (EGFR) activates a MAP kinase cascade that eventually activates ERK. The scaffolding function of FAK brings ERK and calpain 2 into a complex, resulting in phosphorylation of calpain 2. This ERK-mediated phosphorylation leads to activation of calpain 2, which can be counteracted by phosphorylation of calpain 2 by PKA. Active calpain 2 can then cleave talin 1, leading to adhesion complex turnover and cell migration. Except under pathological conditions associated with cell death, such as axonal transection, neurodegeneration and tissue ischemia, the levels of calcium required to activate calpains maximally in vitro do not exist within living cells. This apparent paradox has led researchers towards the idea that other regulatory mechanisms can lower this requirement in vivo. Several different modes of regulation have been identified, although their contributions in vivo have not yet been determined. The large subunits of some calpains are autolyzed on activation, which removes domain I and abolishes the N-terminal link between the large and small subunits, thereby allowing movement of domain II (Baki et al., 1996; Cong et al., 1989; Elce et al., 1997; Guttmann et al., 1997; Imajoh et al., 1986; Molinari et al., 1994;Suzuki and Sorimachi, 1998; Suzuki et al., 1981a). The truncated large subunit is catalytically active and has a lower requirement for calcium (Baki et al., 1996; Imajoh et al., 1986; Suzuki and Sorimachi, 1998; Suzuki et al., 1981b). However, this event is clearly not required for catalytic activity (Cong et al., 1989; Elce et al., 1997; Guttmann et al., 1997; Molinari et al., 1994), which suggests that it functions more in the progression of activation than in its initiation. The binding of phospholipids also decreases the calcium requirement for calpains in vitro (Arthur and Crawford, 1996; Melloni et al., 1996; Saido et al., 1992; Suzuki et al., 1992; Tompa et al., 2001), but the in vivo relevance of this is unknown. Similarly, regulation of protein-protein interactions changes the calcium requirements of calpains (Melloni et al., 2000a; Melloni et al., 2000b;Melloni et al., 1998; Melloni et al., 2000c; Michetti et al., 1991; Salamino et al., 1993), but their roles in activation are not clear. Finally, calpains are regulated by their best-known interacting partner, the endogenous calpain inhibitor calpastatin (Wendt et al., 2004). Although overexpression of calpastatin in cells can decrease calpain activity, escape from calpastatin is not sufficient to activate calpains. Furthermore, structural and

biochemical data indicate that calpastatin might bind preferentially to calciumactivated calpains (Barnoy et al., 1999; Tullio et al., 1999), suggesting that this is an attenuation mechanism rather than a preventive one. Friedrich has recently provided an explanation for this apparent paradox (Friedrich, 2004). He proposes that the calpain system developed this high requirement for calcium during evolution as a safety device to prevent potentially destructive hyperactivity of calpains, and that it is preferable for calpains to work at much less than half-maximal activity. Several pieces of evidence support this idea, including structural considerations, the need for spatial and temporal regulation of calpains and the benefits of a large separation between normal and pathological function. In addition, phosphorylation at several sites controls the activities of calpains. Calpain 2 is activated by phosphorylation of Ser50 by the ERK mitogen-activated protein (MAP) kinase (Glading et al., 2004) during migration of fibroblasts and in keratinocytes stimulated with epidermal growth factor (EGF;Glading et al., 2000; Satish et al., 2005). Phosphorylation of calpain 2 at this site is particularly interesting since calpain 1, which does not contain a phosphorylatable site in this region, does not seem to play a major role in EGF-mediated motility (Glading et al., 2000; Satish et al., 2005). Instead, calpain 1 is important for IP-9-induced motility, which requires intracellular calcium flux (Satish et al., 2005). By contrast, EGFmediated activation of calpain 2 by phosphorylation occurs in the absence of increased calcium levels. Furthermore, calpain 3 has a glutamic acid residue at this position that could act as an activating phosphomimetic, which might explain why calpain 3 lacks a requirement for increased calcium levels. Together, these data suggest that calcium and growth-factor-mediated phosphorylation can independently activate calpains in an isoform-specific fashion. Interestingly, only membraneproximal calpain 2 is activated by ERK-mediated phosphorylation (Glading et al., 2001), which suggests that there are alternative modes of activation for certain calpain 2 subpopulations. Unsurprisingly, the MAP kinase kinase MEKK1 is required for normal calpain 2 activity (Cuevas et al., 2003). MEKK1 associates with focal adhesion kinase (FAK) in adhesion complexes and appears to act upstream of ERK in the regulation of calpain 2 activation and subsequent detachment of the rear of the cell during migration. Note that the adaptor function of FAK mediates the assembly of an ERK-calpain 2 complex at peripheral adhesion sites (Carragher et al., 2003). The formation of this complex and the activity of ERK are both required for normal calpain 2 activity and for processes dependent on calpain 2 such as adhesion complex turnover, transformation and cell migration. There is thus a novel signaling pathway by which growth factors regulate cell migration via phosphorylation-dependent activation of calpains (Fig. 2B). Calpain activity can also be inhibited by phosphorylation. Cyclic-AMP-mediated activation of protein kinase A (PKA) can block EGF-induced activation of calpain 2 and fibroblast migration (Shiraha et al., 2002). This appears to occur through phosphorylation of calpain 2 by PKA, which probably restricts calpain 2 to an inactive conformation (Shiraha et al., 2002; Smith et al., 2003). The residues in calpain 2 (Ser369 and Thr370) that appear to be the PKA targets are conserved in other calpains, which suggests that phosphorylation of domain III represents yet another mechanism for regulating calpain activity.

Calpain substratesAmong the >100 proteins identified as calpain substrates are transcription factors, transmembrane receptors, signaling enzymes and cytoskeletal proteins. Although calpains can lead to extensive degradation of some of these substrates, most are cleaved in a limited fashion, resulting in stable protein fragments that can have functions different from those of their intact forms. Such limited proteolysis might be correlated with a highly specific recognition sequence. However, no single consensus

sequence has been found to have significant value for predicting whether a protein can be proteolyzed by calpains or even where calpains cleave a known substrate. Instead, recognition and proteolysis seem to be controlled by multiple determinants, including but not limited to secondary structure and PEST score (Tompa et al., 2004). This suggests that calpains cleave their substrates in disordered regions between structured domains. Nevertheless, despite this complex set of determinants, there is a significant preference for particular sequences immediately surrounding the site of proteolysis, and studies that have elucidated these preferences have provided valuable tools with which the calpain systems may be specifically and efficiently manipulated (Tompa et al., 2004). One obvious clue as to how calpains might affect cell motility comes from the fact that numerous adhesion complex components and migration-related proteins are substrates for calpains (Table 3) (Glading et al., 2002; Goll et al., 2003). Although proteolysis of most of these adhesion-related substrates has been demonstrated in vitro as well as in cell culture, several issues have made it difficult to determine which are relevant to calpain-mediated pathways in vivo. The specificity and extent of proteolysis of adhesion complex components can vary between cell types (S.J.F. and A.H., unpublished). Further complicating the issue is the fact that most of these substrates can be proteolyzed in vitro equally well by either calpain 1 or calpain 2, which can have widely differing subcellular localizations and cell-type-specific expression patterns even in culture. Recent studies have begun to identify the motility-related substrates that are most readily and consistently cleaved by calpains, as well as the isoforms responsible in living cells. Knockout of calpain small subunit 1 (CSS1 or Capn4) in mice (Arthur et al., 2000) leads to reduced expression and activities of both calpain 1 and calpain 2 (Dourdin et al., 2001). Embryonic fibroblasts isolated from these mice exhibit decreased proteolysis of several reported substrates, including FAK, paxillin, spectrin, cortactin and talin 1. However, others do not appear to be consistently cleaved in mouse embryonic fibroblasts, including vinculin, RhoA, -actinin and Src (Dourdin et al., 2001) (S.J.F. and A.H., unpublished). The relevance of these findings remains to be determined, but, together with other reports, they indicate that some calpain substrates are more readily cleaved than others in certain cell types. However, this cell line cannot reveal which calpain isoform is responsible in each case, since the activities of both calpain 1 and calpain 2 are reduced in these cells. Studies of cells isolated from calpain-1-knockout mice reveal that, despite the absence of calpain 1, these cells can proteolyze many substrates normally, including FAK, paxillin, spectrin and talin 1 (Azam et al., 2001). However, other calpain isoforms could be compensating for calpain 1 deficiency in these cells. More recently, RNA interference (RNAi) technology has been employed to knockdown expression of individual calpain isoforms (Franco et al., 2004a). Interestingly, knockdown of calpain 2 results in decreased proteolysis of FAK, paxillin, spectrin, cortactin and talin 1, while knockdown of calpain 1 has no effect on proteolysis of these proteins (Franco et al., 2004a) (B. Perrin and A.H., unpublished). Therefore, it seems that many motilityrelated proteins require calpain 2 for proteolysis and are either not cleaved by calpain 1 in living cells or are cleaved by compensatory mechanisms in the absence of calpain 1.

Calpains and cell motilityCalpains were first implicated in cell migration by studies showing that pharmacological inhibition of calpains results in reduced integrin-mediated cell migration (Huttenlocher et al., 1997; Palecek et al., 1998). This inhibition leads to stabilization of adhesion complexes and therefore an increase in adhesiveness, thus reducing the rate of detachment of the rear of the cell and decreasing cell migration.

Knockout studies support these findings: embryonic fibroblasts from CSS1-deficient mice display a similar reduction in integrin-mediated motility (Dourdin et al., 2001). Since inhibition of calpains by several means results in formation of large peripheral adhesion complexes, calpains were initially thought to regulate cell motility primarily by destabilizing adhesion to the ECM and promoting rear detachment. However, subsequent studies have demonstrated roles for calpains in many aspects of migration, such as cell spreading, membrane protrusion, chemotaxis, and adhesion complex formation and turnover (Fig. 3). Cell spreading Cell spreading on ECM components is a complex process involving dynamic reorganization of the actin cytoskeleton in response to integrin-mediated signaling through various pathways. Roles for calpains during cell spreading have been demonstrated in several different systems, but these studies do not reveal one clear function for calpains during this process. Inhibition of the primary calpain in platelets, calpain 1, reduces the ability of these cells to spread, possibly by decreasing proteolysis of adhesion complex proteins (Croce et al., 1999). Inhibition of calpains also reduces spreading in T cells, vascular smooth muscle cells and pancreatic cells (Parnaud et al., 2005;Paulhe et al., 2001; Rock et al., 2000). In fibroblasts, overexpression of calpastatin leads to decreased levels of calpain 2 and a decrease in cell spreading and spreading-related actin rearrangements (Potter et al., 1998). This might be caused by an increase in the steady-state levels of the ERM protein ezrin; calpains can proteolyze ezrin and regulate its mRNA levels by an unknown mechanism (Potter et al., 1998). By contrast, spreading of bovine aortic endothelial (BAE) cells is reported to depend specifically on calpain 1, since overexpression of calpain 1 leads to increased cell spreading and a dominantnegative calpain 1 reduces spreading (Kulkarni et al., 1999). However, in the same cell type, calpain 1 can proteolyze RhoA, thereby generating a dominant-negative fragment that inhibits cell spreading (Kulkarni et al., 2002). Studies showing that calpain 1 is important for the formation of early clusters of adhesion molecules that might be sites of Rac1 activation in the early stages of spreading in BAE cells support the idea that calpain 1 positively regulates spreading (Bialkowska et al., 2000). By contrast, knockdown of calpain 1 in several fibroblast cell lines does not affect the ability of these cells to spread (Franco et al., 2004a). Further complicating the issue is the fact that inhibition of calpains in neutrophils might increase spreading of these cells (Lokuta et al., 2003). CSS1 also appears to play a role in cell spreading through its interaction with the guanine nucleotide exchange factor (GEF) PIX (Rosenberger et al., 2005). PIX binds to and colocalizes with calpains in small integrin-containing clusters during the early stages of cell spreading in CHO-K1 cells. Treatment of these cells with calpain inhibitors reduces spreading, which can be overcome by overexpression of PIX. Interestingly, an PIX mutant that cannot bind CSS1 does not rescue the spreading defect, but a GEF-deficient PIX mutant does. PIX therefore appears to have a GEFindependent role in cell spreading downstream of calpains.



Fig. 3. Motility-related processes known to be affected by calpains and the substrates or binding partners acting as effectors. Calpain 2 can cleave adhesion complex proteins such as FAK, paxillin and talin 1, possibly resulting in integrin activation, adhesion complex turnover or detachment of the cell rear. Proteolysis of the actin-regulating protein cortactin might lead to inhibition of membrane protrusion. Cleavage of integrin -tails might be important for the formation of small integrin clusters during the early stages of cell spreading, whereas proteolysis of the small GTPase RhoA negatively regulates cell spreading. Interaction of PIX with calpain small subunit 1 (CSS1) can also mediate cell spreading. Proteolysis of the adaptor protein MARCKS might also regulate cell migration in myoblasts, possibly by promoting adhesion formation. The isoforms required for proteolysis of integrins, RhoA and MARCKS remain to be determined, as do the processes affected by proteolysis of nearly 100 other calpain substrates. Membrane protrusion Many studies of calpains and cell spreading also suggest that calpains regulate actinbased mechanisms involved in membrane protrusion. Inhibition of calpains by calpastatin or pharmacological inhibitors leads to formation of abnormal lamellipodia and filopodia (Potter et al., 1998). Likewise, embryonic fibroblasts from CSS1knockout mice exhibit altered morphologies, displaying thin membrane projections (Dourdin et al., 2001). These cells also exhibit global increases in transient membrane protrusiveness and faster and more-frequent, but less-stable, leading edge protrusions (Franco et al., 2004a). Calpain 2 appears to be the isoform responsible, since knocking it down reproduces the protrusion defects of CSS1deficient cells. The actin-regulatory protein cortactin is a calpain substrate and probably an important downstream target of calpain 2 in the regulation of membrane protrusions (B. Perrin and A.H., unpublished), because expression of a calpainresistant form of cortactin leads to membrane defects similar to those seen in calpain-2-knockdown cells. Further support for calpains negatively regulating membrane protrusion comes from studies showing that calcium transients in filopodia of neuronal growth cones act through calpains to reduce lamellipodial protrusion (Robles et al., 2003). Chemotaxis Calpains also negatively regulate membrane protrusion in neutrophils. High levels of calpain activity exist in resting neutrophils, and inhibition of these enzymes promotes membrane protrusion and rapid chemokinesis (Lokuta et al., 2003). This contrasts with most other cell types, in which calpain inhibition reduces cell migration. In cell types in which calpains inhibit cell migration, the underlying mechanism might involve negative regulation of the Rho GTPases Cdc42 and Rac1, since calpain inhibition promotes activation of Cdc42 and Rac in neutrophils. The effects are comparable with treatment with chemoattractants such as N-formyl-methionyl-luecylphenylalanine (fMLP), which increase chemokinesis (Lokuta et al., 2003). Interestingly, whereas inhibition of calpains promotes random migration of neutrophils, it reduces the directional migration of neutrophils up a gradient of chemoattractant (Lokuta et al., 2003). Spatial regulation of calpain activity might therefore be required for optimum chemotaxis of neutrophils, and calpains could play a role in directional sensing or cell polarization during directed cell migration. Adhesion complex regulation Because dynamic regulation of adhesion to the ECM is required for cell migration, the mechanisms by which adhesion complexes are formed and subsequently disassembled are key to cell motility. For some time, inhibition of calpains has been known to alter the morphology and stability of adhesion complexes, but only now are we beginning to elucidate the details of calpain-mediated regulation of adhesion

complexes. Although a role for calpains in the disassembly of adhesion sites has been well documented, whether calpains are important for the formation of adhesion complexes remains unclear. As previously mentioned, calpains appear to be important for induction of small, integrin-containing protein clusters at the early stages of spreading (Bialkowska et al., 2000; Bialkowska et al., 2005). However, these clusters do not seem to be precursors of typical adhesion complexes; so their significance is not known. Calpain-mediated proteolysis of talin 1 might be involved in assembling adhesion complexes, since proteolysis of talin 1 by calpains promotes its binding to integrin -tails, which is known to be crucial for inside-out activation of integrins (Calderwood, 2004; Calderwood et al., 2002; Calderwood et al., 1999; Yan et al., 2001). Proteolysis of the actin-binding protein myristoylated alanine-rich protein kinase C substrate (MARCKS) might also play a role in the formation of adhesion complexes, since inhibition of calpains in myoblasts leads to defects in new adhesion formation and migration coincident with an accumulation of MARCKS (Dedieu et al., 2004). However, several lines of evidence indicate that adhesion complexes can form normally when calpain activity is reduced; so calpains do not appear to be required for assembly of adhesion complexes in most cell types. As discussed above, calpains can cleave many adhesion complex proteins and downregulation of calpain activity results in large adhesion complexes and inhibits cell detachment. Calpains could therefore be important for destabilization/disassembly of adhesion complexes. Inhibition of calpains by calpastatin or pharmacological agents blocks microtubule-mediated turnover of adhesion complexes after nocodazole washout. This suggests that calpains act downstream of microtubules to mediate adhesion complex disassembly (Bhatt et al., 2002). Knockdown of calpain 2 by RNAi slows the rate at which adhesion complexes disassemble, leading to formation of large, elongated adhesion complexes (Franco et al., 2004b). Furthermore, expression of a calpain-resistant talin 1 mutant in talin-1null cells also decreases adhesion complex disassembly rates. This indicates that calpain-2-mediated proteolysis of talin 1 regulates adhesion turnover. How talin 1 proteolysis results in adhesion disassembly is not known, but it is likely that this affects both the structural and signaling functions of talin 1 within adhesion complexes. Since talin 1 is cleaved more readily than most other calpain 2 substrates (S.J.F. and A.H., unpublished), its proteolysis might represent the major mode of calpain-2-mediated adhesion disassembly. Future studies will have to determine whether proteolysis of other substrates is also involved.

Calpains in human diseaseCalpains have been connected to a variety of pathological conditions (Zatz and Starling, 2005), including stroke and ischemia (Vanderklish and Bahr, 2000), susceptibility to non-insulin diabetes mellitus (Horikawa et al., 2000) and in the pathogenesis of muscular dystrophies (Ono et al., 1998; Sorimachi et al., 2000). Calpain activity appears to play a central role in the movement of immune cells (Lokuta et al., 2003; Stewart et al., 1998), thereby participating in the development of inflammation in normal and pathological conditions such as chronic inflammatory disease (Cuzzocrea et al., 2000; Shields and Banik, 1998; Shields et al., 1998). Furthermore, calpain 2 expression is upregulated in some cancers and has recently been associated with disease progression in patients with breast cancer (Rios-Doria et al., 2003; Wang et al., 2005;Carragher et al., 2004; Huber et al., 2004). The coordinate regulation of adhesion structures by calpains and Src tyrosine kinases places calpains in a crucial role at the interface of kinase and protease cascades that regulate migration of tumor cells and their invasive properties (Carragher et al., 2001;Carragher and Frame, 2002; Carragher et al., 2002; Mamoune et al., 2003). Pathways involving calpains might thus represent an attractive therapeutic target. Future investigations should delineate whether information about the roles of

calpains in motility can facilitate development of drugs to treat a variety of human diseases, including cancer and chronic inflammatory disease.

Conclusions and perspectivesLimited proteolysis by calpains has emerged as a key signal-transducing mechanism that probably functions at the interface of integrin- and growth-factor-mediated signaling to regulate cell migration. The regulation of calpains by calcium, phosphoinositides, and phosphorylation by MAP kinase and PKA pathways places calpains at the center of different signaling pathways controlling many basic cellular processes in addition to cell motility. Crucial to calpain function is the tight regulation of its proteolytic activity, which must be both temporally and spatially controlled during cell migration. Substantial evidence suggests that calpains are activated in a highly localized manner and can be targeted to discrete regions within the cell. However, despite recent progress, our understanding of calpain function during cell migration remains limited. This might be partly attributed to the number and diversity of calpain isoforms; of the 16 known calpain isoforms, only calpain 1, calpain 2 and CSS1 have been studied with respect to migration. Furthermore, there are >100 substrates that can be cleaved by calpains, which makes it difficult to dissect how calpains orchestrate their effects during cell migration. Generation of calpain-resistant substrates will provide important mechanistic clues, but it is important to consider that calpains, like Src-mediated signaling pathways, probably operate by targeting multiple substrates to modify specific stages of the cell motility cycle. Establishing the details involved will require the combination of sophisticated imaging and proteomics-based approaches in both in vitro and in vivo systems.

Calpain activity and muscle wasting in sepsisABSTRACTMuscle wasting in sepsis reflects activation of multiple proteolytic mechanisms, including lyosomal and ubiquitin-proteasome-dependent protein breakdown. Recent studies suggest that activation of the calpain system also plays an important role in sepsis-induced muscle wasting. Perhaps the most important consequence of calpain activation in skeletal muscle during sepsis is disruption of the sarcomere, allowing for the release of myofilaments (including actin and myosin) that are subsequently ubiquitinated and degraded by the 26S proteasome. Other important consequences of calpain activation that may contribute to muscle wasting during sepsis include degradation of certain transcription factors and nuclear cofactors, activation of the 26S proteasome, and inhibition of Akt activity, allowing for downstream activation of Foxo transcription factors and GSK-3. The role of calpain activation in sepsisinduced muscle wasting suggests that the calpain system may be a therapeutic target in the prevention and treatment of muscle wasting during sepsis. Furthermore, because calpain activation may also be involved in muscle wasting caused by other conditions, including different muscular dystrophies and cancer, calpain inhibitors may be beneficial not only in the treatment of sepsis-induced muscle wasting but in other conditions causing muscle atrophy as well. muscle proteolysis; calcium; atrophy; calpastatin seen in patients with sepsis (44, 55). Several other catabolic conditions, such as burn injury, cancer, uremia, and AIDS, are also associated with muscle wasting (20, 27, 29, 79). Muscle wasting has severalLOSS OF MUSCLE MASS IS COMMONLY

significant clinical consequences. For example, loss of muscle mass results in weakness and fatigue, in turn resulting in delayed ambulation and prolonged rehabilitation. When patients are bedridden for long periods of time, the risks for thromboembolic events as well as for pneumonia and other pulmonary complications increase. Prolonged bed rest in itself causes loss of muscle mass, thus creating a vicious cycle (35). Patients treated in the intensive care unit may need ventilatory support for extended periods of time when respiratory muscles are atrophying (82). Under normal conditions, muscle mass is maintained by a balance between protein synthesis and degradation, and muscle wasting can occur in any situation when this equilibrium is perturbed. There is evidence that loss of muscle mass during sepsis to a great extent reflects activated breakdown of muscle proteins, in particular the contractile proteins actin and myosin (48), but reduced protein synthesis may also contribute to sepsis-induced muscle wasting (62). Although increased expression and activity of the ubiquitin-proteasome proteolytic pathway, including a dramatic upregulation of the muscle-specific ubiquitin ligases atrogin-1 and MuRF1 (16, 40), play an essential role in sepsis-induced muscle wasting (33, 113), other proteolytic mechanisms are also involved (50). For example, recent studies suggest that autophagy (67, 121) and lysosomal enzymes, in particular cathepsin L (26, 63), as well as certain peptidases, such as tripeptidyl peptidase II (115), are activated in skeletal muscle during sepsis. The purpose of the present review is to discuss evidence that activation of the calpain system is an additional important mechanism of sepsis-induced muscle wasting. Although calpains may be activated secondary to cell injury and calcium leak, there is increasing evidence that calpain activity is also regulated by physiological and pathophysiological mechanisms in intact cells (39). Certain aspects of the role of calpains in muscle wasting were discussed recently by others (9, 22) and in a review from our laboratory (49). The present review adds to previous information by focusing mainly on the calpain system in sepsis-induced muscle wasting and by discussing some of the controversy that exists with regards to the relative importance of calpain- and caspase-dependent proteolysis in catabolic conditions. It also highlights pertinent recent reports on the role of calcium and calpains (4, 6, 15, 25, 33, 75, 83, 89, 92, 99, 104, 111) published after our previous review (49). Other aspects of the regulation of muscle mass during sepsis, including the role of transcription factors and nuclear cofactors and the influence of posttranslational modifications of these regulators, were reviewed recently (45, 46, 49). Various mechanisms by which calcium and calpain activity may regulate muscle mass and that are discussed in the present review are shown in Fig. 1. It should be noted that although the present review is focused on the role of calpain activation in skeletal muscle, there is evidence that the calpain system may be involved in the regulation of protein balance in the cardiomyocyte as well (15, 37).

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Fig. 1. Summary of the role of calpain activation in sepsis-induced muscle wasting. Uptake and cellular concentrations of calcium are increased in skeletal muscle during sepsis (34). Calpains are activated by calcium (39), although other mechanisms may also contribute to calpain activation. The most important muscle wasting-related consequence of calpain activation is cleavage of myofibrillar cytsokeletal proteins resulting in disruption of the sarcomere and release of myofilaments that are subsequently ubiquitinated and degraded by the 26S proteasome (47, 52, 93, 94). In addition, calpain activation may regulate the degradation of various transcription factors involved in muscle wasting (42, 66, 111). Calpains may regulate the 26S proteasome activity (69, 92), and it is possible that calcium can activate the 26S proteasome through other mechanisms as well (69). Finally, recent studies (92) suggest that calpain activation in skeletal muscle results in inhibited Akt activity, which in turn results in activation of Foxo transcription factors and GSK-3 (stimulating muscle proteolysis) and inactivation of mammalian target of rapamycin (mTOR), inhibiting protein synthesis. The Calpain System Calpains are nonlysosomal, calcium-dependent cysteine proteases. The calpains constitute a family of at least 14 members that are ubiquitous enzymes, such as and m-calpain, or tissue specific, such as the muscle-specific calpain 3, also called CAPN3 or p94. The - and m-calpains are heterodimers composed of two subunits of 80 and 30 kDa, respectively. The larger subunit contains the catalytic domain, whereas the smaller unit has regulatory functions. The calpain system, its various members, as well as regulatory mechanisms were reviewed extensively elsewhere (39). It should be noted that the role of p94 is probably unique and different (or even opposite) to that of the ubiquitous - and m-calpains. For example, whereas the expression and activity of - and m-calpain are increased in many muscle-wasting conditions, p94 is typically not affected or is even decreased. A striking example of the opposite role of p94 in muscle compared with the role of - and m-calpain is the observation that certain types of muscular dystrophy are caused by p94 deficiency (57, 84, 102). Certain other muscle atrophy conditions (experimental cancer cachexia and muscle denervation) are also associated with the downregulation of p94 (17, 96). Of note, sepsis-induced muscle wasting may be unique with regards to the regulation of p94. For example, in previous studies (34, 112), we observed increased p94 mRNA levels in muscle from septic rats, although we have not found evidence of increased p94 activity in septic muscle. Interestingly, the activity of - and m-calpain may be affected by p94 since p94 is able to cleave calpains (and calpastatin) (39). In the present review, we will mainly discuss the role of - and m-calpain in sepsisassociated muscle wasting. The regulation of calpain activity is complex. Calpains are typically in an inactive state under basal conditions. Calcium is the most important activator of calpains. Binding of calcium results in conformational changes, allowing for the catalytic site of the molecule to become activated. In addition to calcium, other factors may also influence calpain activity. For example, recent studies (39) suggest that - and mcalpain activity can be influenced by phosphorylation. Other studies (86, 90, 120) suggest that certain phospholipids, in particular phosphatidylinositol, influence calpain activity by various mechanisms, including lowered calcium concentration needed for autolysis of - and m-calpain. Additional molecules have also been found to regulate calpain activity by lowering the calcium requirements for activation, including isovalerylcarnitine (80) and a 4045 kDa endogenous "activator" present in skeletal muscle (81). An additional important regulator of calpain activity is the endogenous inhibitor calpastatin (39). Of note, calcium not only regulates the activity of calpains but also influences the binding of calpastatin to calpain, resulting in inhibited calpain activity.

In addition, calpain is autocatalyzed, i.e., activated calpain degrades itself. Activated calpain can also degrade calpastatin, adding an additional level of complexity to the regulation of the calpain system. Muscle Wasting is Associated with Increased Calcium Uptake and Concentrations Multiple previous studies (36) provided evidence for a role of calcium-dependent mechanisms (probably at least in part reflecting calpain activation) in muscle wasting. In early studies (5), treatment of incubated muscles with calcium or a calcium ionophore increased protein degradation. In recent experiments (69) in our laboratory, treatment of cultured myotubes with the calcium ionophore A23187 [GenBank] stimulated proteasome activity and this effect was at least in part regulated by calpain activation. In other reports, muscle calcium uptake and intracellular concentrations were increased in sepsis (11, 34) as well as in other catabolic conditions, including burn injury (88), and cancer (21). Experiments using the "calcium antagonist" dantrolene, a drug that blocks the net release of calcium from the sarcoplasmic reticulum into the sarcoplasm (59), as well as recent (unpublished) observations in our laboratory using dexamethasone-treated myotubes, suggest that increased calcium levels in atrophying muscle reflects increased store operated calcium entry. Taken together, studies showing calciumdependent regulation of muscle protein degradation provide important indirect evidence for a role of calpains in muscle wasting (although, of course, calcium may regulate protein degradation through other mechanisms as well). Additional evidence for a role of calcium and calcium-regulated activation of calpains in muscle proteolysis was provided in a recent study by Smith and Dodd (92). In that study, incubated muscle preparations from rats were treated in vitro with 3.5 mM calcium in the absence or presence of the calpain inhibitor calpeptin. Exposing the muscles to calcium resulted in calpain activation, assessed by determining tissue levels of the 190 kDa talin cleavage fragment, and a 65% increase in protein degradation. Treatment of the muscles with calpeptin prevented calpain activation and the increase in protein degradation, providing evidence for a link between calcium-regulated calpain activation and muscle proteolysis. Calpain Activity is Increased in Skeletal Muscle During Sepsis and May at Least in Part be Caused by Inhibition of Calpastatin Activity In previous studies (34, 112), increased calcium levels and upregulated expression of calpains in skeletal muscle during sepsis provided indirect evidence of calpain activation. More direct evidence of increased calpain activity and expression in skeletal muscle during sepsis was reported by Bhattacharyya et al. (13) and Voisin et al. (105). In the study by Bhattacharyya et al. (13), calpain activity, determined by measuring the degradation of the calpain substrate azocasein in muscle extracts, was increased by 70% in rats made septic by intraabdominal implantation of pellets containing Escherichia coli and Bacteroides fragilis bacteria. In the study by Voisin et al. (105), "chronic sepsis" 6 days after the intravenous injection of live E. coli bacteria was associated with an 1.5-fold increase in mRNA levels for m-calpain (-calpain mRNA levels were not determined) and a tendency (although not statistically significant) for increased lysosomal and calcium-dependent protein breakdown determined by using the cysteine protease inhibitor E-64c. In a recent study (110) in our laboratory, the degradation of different calpain-specific substrates was increased in muscle extracts from septic rats, consistent with sepsis-induced increase of calpain activity. Because, in the same study, - and m-calpain activity was not increased in septic muscle when measured by zymography (a method in which calpains are separated from calpastatin), increased net calpain activity in muscle extracts (containing both calpains and calpastatin) may represent reduced calpastatin activity. This was indeed confirmed in the same study when calpastatin activity was measured separately and was reduced by 4060% in muscles from septic rats. In more recent experiments (33) in our laboratory, transfection of cultured muscle cells with a plasmid expressing calpastatin cDNA resulted in elevated cellular calpastatin levels and reduced protein

degradation in dexamethasone-treated muscle cells. The use of dexamethasonetreated muscle cells in those experiments was important because glucocorticoids are important mediators of muscle proteolysis during sepsis (43). The observation that reduced calpastatin activity may be a mechanism of calpain activation in skeletal muscle during sepsis (33, 110) is in line with a recent report by Tidball and Spencer (103). In their study, transgenic overexpression of calpastat

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